Modeling of a bioterrorist attack with Francisella tularensis by WHO and CDC showed that the extreme infectivity of the pathogen would lead to very high economic costs for society. Considering the high morbidity of a bioterrorist attack with F. tularensis, there is a need to develop an effective response to such an event. Since bioterrorist attacks are unpredictable and tularemia is a rapidly progressive disease, the effects of post-exposure antibiotic treatment might be marginal at best. In contrast, the availability of a safe and effective vaccine would be a true prophylactic measure. Several attenuated Francisella tularensis strains were developed in the 1950's and proven to be efficacious as live vaccines in humans. The most notable success was the live vaccine strain, F. tularensis LVS, which has been given to tens of thousands of individuals and until recently had an IND status in the US. However, since the mechanism(s) of the attenuation of F. tularensis LVS and other live vaccine strains used in the former Soviet Union are unknown, they most likely will not be licensed for widespread use in humans. Given the relative efficacy of these vaccines, together with the availability of recently developed genetic techniques, it should be possible to develop a new generation of F. tularensis vaccines based on specific mutations that confer attenuation. This is the focus of the present proposal. Although F. tularensis LVS has been widely used and shown to be efficacious for prevention of laboratory-acquired tularemia, recent work using an experimental mouse model indicated that it affords suboptimal protection against aerosol exposure to the highly virulent subsp. tularensis (type A). This lack of efficacy is not entirely surprising since F. tularensis LVS was developed by attenuation of a Russian strain belonging to the other clinically important, but less virulent subspecies, holarctica (type B F. tularensis). Recently, we showed that subsp. tularensis contains approximately 30 genes that are missing in subsp. holarctica. Since it is possible that these include additional protective antigens, the retention of these genes in future attenuated vaccines could be advantageous. Recent work on a spontaneous mutant of Schu S4 supports this hypothesis. Thus, we will use the virulent F. tularensis Schu S4 strain of subsp. tularensis as the basis for generating attenuated vaccines. In this regard, recent analyses of genomic F. tularensis sequences have revealed that targets widely used for attenuating other bacterial pathogens are present. For example, all of the enzymes of the shikimate pathway and the purine nucleotide pathway are present in the Schu S4 strain. Furthermore, we recently developed a genetic system that allows the creation of specific mutants of F. tularensis Schu S4 by use of allelic replacement. Armed with this background data and information, the specific aims of this proposal are: To optimize genetic systems for use in F. tularensis subsp. Tularensis. To identify targets for attenuation. To screen for virulence of gene-deficient bacteria in vitro and in vivo To use attenuated strains for immunization of mice and assay for humoral and cell-mediated immunity To determine protective efficacy of mutants in immunized mice against aerosol challenge with virulent F. tularensis subsp. tularensis strains. To determine correlates of protection.